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'''This sandbox is in use until August 1, 2011 for UMass Chemistry 423. Others please do not edit this page. Thanks!'''

Introduction
Neuraminidase protein is one of two glycoproteins that coats the envelope of the influenza virus. Neuraminidase's particular function is the removal of sialic acid from the host cell, allowing the replicated influenza viruses to escape the host cell and spread to other cells.

Tamiflu, a drug designed to combat influenza, binds to and inhibits the function of neuraminidase, disabling its function and consequently disallowing the influenza virus to spread between cells.

Influenza is a disease caused by the various species and genera of the influenza RNA virus. The disease, commonly referred to as "the Flu," is highly contagious and travels around the world in seasonal epidemics. It is responsible for the deaths of between 250,000 and 500,000 people each year, and these numbers can sometimes reach the millions in particularly pandemic years. The virus is particularly lethal to people with weakened immune systems, since the virus can enable the spread of dangerous secondary infections such as pneumonias within the host.

The typical symptoms of the Flu include chills, fever, a sore throat, muscle pains, severe headache, coughing, and weakness or fatigue, as well as general discomfort.

Overall Structure
Influenza (flu) Neuraminidase is a homotetramer, with four identical subunits. Each subunit consists mostly of antiparallel beta sheets and three alpha helices, with a beta-propeller folding pattern (Secondary Structure ). As shown, the alpha helices lie toward the center of the protein.

The Active Site is a pocket on the surface of the protein, lined with highly conserved residues. Shown here is the ligand Oseltamivir (Tamiflu) bound to one of the four subunits. When Tamiflu is bound, it tightly blocks the virus release sites on nonresistant strains of flu neuraminidase because it binds with the hydrophilic residues exposed on the surface.

Different conformations account for drug resistance of flu neuraminidase. 2hu4 is the “closed” conformation of the wild type 3hu0. For example, a mutation at the site H274Y, shown here on the closed-form wild type, causes a change in the usual binding site for Tamiflu, possibly affecting resistance.

Research on the structures of the different mutants of flu Neuraminidase suggests that the slight change in the binding site affects the drug resistance of the molecule. In a specific case presented by Wang, et al., the H5N1 mutant being observed has a hydrophobic Tyr347 compared to the hydrophilic Asn347 of the wild type and closed form. Since Tamiflu binds to hydrophilic regions of the pore, this change has the potential to greatly affect the binding affinity, hence the mutant’s resistance to oseltamivir.

Drug Binding Site
Flu neuraminidase is a homotetramer, and each of the four protein chains has a catalytic site. The catalytic sites are shown here with Tamiflu shown in purple.

Tamiflu was designed specifically to fit to the binding site of neuraminidase by induced fit. The binding site contains a loop of residues 147-152. When it binds at the active site, Tamiflu pulls a loop made of Asp151 and Glu119 residues closer to the inhibitor, thereby enclosing the Tamiflu inhibitor.

There are five specific residues that are in contact with the Tamiflu substrate - Arg292, Glu276, Arg152, Arg371, and Arg118. They are shown here in the active site.

Tamiflu does not work on all types of the flu virus, and some are becoming immune to it. However, it was recently discovered that one of the subunits has a larger surface-accessible cavity in the substrate binding region. The larger end of this cavity is not in contact with Tamiflu when the substrate is bound. This presents an opportunity to design another drug with greater specificity to this pocket.

Additional Features
Neuraminidase breaks the sialic acid groups from glycoproteins. Hemagglutinin from on the virus attaches to the sialic acid groups. The virus is from the cell and is released when neuraminidase breaks the sialic acid groups. This will allow flu replication to happen and a neuraminidase inhibitor, such as Tamiflu, is required to stop neuraminidase. The interaction of Tamiflu with N1 neuraminidase is shown with this morph (1). Tamiflu in this model is bound when it is visible and the loop in moved in towards the drug through induced fit.

When neuraminidase breaks the sialic acid groups, the virus (virion) is released. This will allow replication of the virus. The inhibitor prevents the breaking of the sialic acid groups, so no virion is released and replicated.

Credits
Introduction - Mike Reardon

Overall Structure - Rachael Jayne

Drug Binding Site - Jackie Dorhout

Additional Features - Nick DeGraan-Weber